WO1987002060A1 - Polypeptides semblables aux facteurs de stimulation de colonies de granulocytes-macrophages (gm-csf) et leurs procedes de production en quantites importantes dans des cellules microbiennes - Google Patents

Polypeptides semblables aux facteurs de stimulation de colonies de granulocytes-macrophages (gm-csf) et leurs procedes de production en quantites importantes dans des cellules microbiennes Download PDF

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WO1987002060A1
WO1987002060A1 PCT/US1986/002106 US8602106W WO8702060A1 WO 1987002060 A1 WO1987002060 A1 WO 1987002060A1 US 8602106 W US8602106 W US 8602106W WO 8702060 A1 WO8702060 A1 WO 8702060A1
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csf
hgm
coli
polypeptide
polypeptides
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PCT/US1986/002106
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English (en)
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John Delamarter
Joachim F. Ernst
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Biogen N.V.
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Publication of WO1987002060A1 publication Critical patent/WO1987002060A1/fr
Priority to DK282887A priority Critical patent/DK282887A/da

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/53—Colony-stimulating factor [CSF]
    • C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to human granulocyte- macrophase colony stimulating factor-like polypeptides (hGM-CSF), DNA sequences, recombinant DNA molecules and processes for producing hGM-CSF. More particularly, the invention relates to hGM-CSF-like polypeptides with a high specific activity, and DNA sequences, recombinant DNA molecules and processes that permit the production of hGM-CSF-like polypeptides in high yields in microbial cells.
  • hGM-CSF human granulocyte- macrophase colony stimulating factor-like polypeptides
  • Granulocyte-Macrophage Colony Stimulating Factor is one of four classes of hemopoietic growth factors known as colony stimulating factors ("CSF's").
  • CSF's colony stimulating factors
  • GM-CSF is a growth factor which regulates the proliferation and differentiation of multipoten- tial cells [E. R. Stanley and P. T. Jubinsky, "Factors Affecting the Growth and Differentiation of Haemo- poietic Cells in Culture," Clinical He atology, 13, pp. 329-48 (1984)].
  • GM-CSF has also been shown to stimulate the formation of clones of neutrophilic granulocytes and mononuclear phagocytic cells from ! -2- single bone marrow cells in vitro [A.
  • the GM-CSF's of this invention should • be useful in the recovery of white blood cells after irradiation or chemotherapy. They also should acti ⁇ vate white blood cells to combat infections of bacteria, fungi, and parasites and to accelerate the maturation of leukemic cells and thereby stop the regeneration of leukemic cells.
  • Mouse GM-CSF also known as mouse CSF-2
  • mouse CSF-2 a 24-26,000 molecular weight glycoprotein which con ⁇ tains no subunits
  • endotoxin- injected mouse lung-conditioned medium [A. . Burgess, supra; N. A. Nicola et al., J. Biol. Chem., 254, pp. 5290-99 (1979)]. It is unable to stimulate the formation of colonies of erythr ⁇ id, eosinophil, mega- karyocyte cells or of T-and B-lymphocytes in suitable culture conditions, which implies that it is highly selective in its proliferative effects on hemopoietic cells [A. W. Burgess and D.
  • Human GM-CSF is also a glycoprotein (24-26,000 daltons). It has been cloned and the cDNA sequence reported [G. G. Wong et al., "Human GM-CSF: Molecular Cloning of the Complementary DNA and Purification of the Natural and Recombinant Proteins," Science, 228, pp. 810-15 (1985)]. Animal cells transfected with this cDNA sequence synthesize glycosylated GM-CSF on the order of l ⁇ g/ml [Wiesbart et al., "Human Granulocyte-Macrophage Colony-Stimu ⁇ lating Factor is a Neutrophil Activator," Nature, 314, pp. 361-63 (1985); Wong, supra] . Therefore, these animal cells have not been able to produce human GM-CSF in sufficient quantities and with the necessary purity for biological and clinical use.
  • the present invention solves the problems referred to above by providing DNA sequences that code for human GM-CSF-like polypeptides and by expressing those sequences in high yields in appro ⁇ priate microbial hosts to produce efficiently and economically large quantities of polypeptides dis ⁇ playing a granulocyte and macrophage colony-stimu ⁇ lating activity. According to this invention, it is possible to modify the amino (5') terminal end of a DNA sequence coding for hGM-CSF and thereby to pro- prise hGM-CSF-like polypeptides in high yields in microbial hosts.
  • the present invention pro ⁇ vides unglycosylated hGM-CSF-like polypeptides unexpectedly having a specific activity of at least
  • the polypep ides of this invention may be used either as produced or after further deriya- tization or modification, against bacterial, fungal or parasitic infections, in the regeneration of leukocytes after irradiation or chemotherapy, as well as in methods and compositions for the treat ⁇ ment of leukemia.
  • These compounds may also be used to reduce the likelihood of opportunistic infections in immunologically compromised individuals, such as those suffering from AIDS.
  • GM-CSF-like polypeptides are administered to the AIDS patients to increase their white blood cell count so as to prevent opportunistic infections, thus lengthening the life and reducing the expense of treating the AIDS patient.
  • Figure 1 depicts the nucleic acid and de ⁇ quizd amino acid sequence for an hGM-CSF DNA as iso ⁇ lated from the U937 cell line.
  • the cleavage site for the signal peptide is indicated by an arrow.
  • Nucleotide 356 is either T or C in the Mo-cell derived cDNA.
  • Figure 2 depicts the nucleic acid sequence for a hGM-CSF cDNA as isolated from the 5637 cell line. The cleavage site for the signal peptide is inciated by an arrow.
  • Figure 3 depicts a restriction endonuclease map of mouse GM-CSF cDNA, wherein both the coding region, and the nick-translated probe which was used to screen the U937 hGM-CSF library are indicated.
  • Figure 4 depicts the construction of the E.coli expression vector pPLmuGM-CSF (p210*) for high level hGM-CSF production in microbial cells in accordance with this invention.
  • Figure 5a and 5b depict schematic repre ⁇ sentation of the E.coli expression vectors (p210* and pCI857, respectively) used to produce hGM-CSF in bacterial cells through a two-plasmid system and their construction through intermediate plasmids.
  • the synthetic region used to replace the 5' coding sequences of hGM-CSF is indicated by a hatched region.
  • Figure 6 depicts the nucleotide sequence and deduced amino acid sequence of the synthetic linker used for E.coli expression (Ncol-Hgal) .
  • Figure 7 depicts the construction of the E.coli single plasmid expression vector p241-48.
  • Figure 8 depicts the yeast alpha mating factor fusion to the hGM-CSF coding region.
  • Figures 9-10 are a schematic representation of the construction of the yeast expression vector p528/l for hGM-CSF production.
  • Nucleotide A monomeric unit of DNA or RNA consisting of a sugar moiety (pentose), a phosphate, and a nitrogenous heterocyclic base.
  • the base is linked to the sugar moiety via the glycosidic carbon (I* carbon of the pentose) and that combination of base and sugar is called a nucleoside.
  • the base characterizes the nucleotide.
  • the four DNA bases are adenine ("A”), guanine (“G”), cytosine ("C”), and thy ine (“T”).
  • the four RNA bases are A, G, C, and uracil ("U”).
  • DNA Sequence A linear array of nucleotides connected one to the other by phosphodiester bonds between the 3' and 5' carbons of adjacent pentoses. ⁇ i
  • Codon A DNA sequence of three nucleotides
  • a triplet which encodes through mRNA an amino acid, a translation start signal or a translation termina- tion signal.
  • the nucleotide triplets TTA, TTG, CTT, CTC, CTA and CTG encode for the amino acid leucine ("Leu"), TAG, TAA and TGA are transla ⁇ tion stop signals and ATG is a translation start signal.
  • Reading Frame The grouping of codons during the translation of mRNA into amino acid sequences. During translation the proper reading frame must be maintained. For example, the DNA sequence GCTGGTTGTAAG may be expressed in three reading frames or phases, each of which affords a different amino acid sequence:
  • Polypeptide A linear array of amino acids connected one to the other by peptide bonds between the ⁇ -amino and carboxy groups of adjacent amino acids.
  • Genome he entire DNA of a cell or a virus. It includes inter alia the structural gene coding for the polypeptides of the substance, as well as operator, promoter and ribosome binding and interac ⁇ tion sequences, including sequences such as the Shine- Dalgamo sequences.
  • Gene A DNA sequence which encodes through its template or messenger RNA (“mRNA”) a sequence of amino acids characteristic of a specific polypeptide.
  • mRNA messenger RNA
  • Transcription The process of producing mRNA from a gene or DNA sequence.
  • Translation The process of producing-a polypeptide from mRNA.
  • Expressio he process undergone by a gene or DNA sequence to produce a polypeptide. It is a combination of transcription and translation.
  • Plasmid A nonchromosomal double-stranded DNA sequence comprising an intact "replicon" such that the plasmid is replicated in a host cell. When the plasmid is placed within a unicellular organism, the characteristics of that organism may be changed or transformed as a result of the DNA of the plasmid. For example, a plasmid carrying the gene for tetra- cycline resistance (TET ) transforms a cell previously sensitive to tetracycline into one which is resistant to it. A cell transformed by a plasmid is called a "transformant”.
  • Phage or Bacteriophage Bacteriophagerial virus, many of which consist of DNA sequences, encapsidated in a protein envelope or coat ("capsid").
  • Cloning Vehicle A plasmid, phage DNA, cosmid or other DNA sequence which is able to repli- cate in a host cell, characterized by one or a small number of endonuclease recognition sites at which such DNA sequences may be cut in a determinable fashion without attendant loss of an essential bio ⁇ logical function of the DNA, e.g., replication, pro- duction of coat proteins or loss of promoter or binding sites, and which contains a marker suitable for use in the identification of transformed cells, e.g., tetracycline resistance or ampicillin resist ⁇ ance.
  • a cloning vehicle is often called a vector.
  • Recombinant DNA Molecule or Hybrid DNA A molecule consisting of segments of DNA from different genomes which have been joined end-to-end outside of living cells and able to be maintained in living cells. i -8-
  • Expression Control Sequence A sequence of nucleotides that controls and regulates expression of genes when operatively linked to those genes. They include the lac system, the ⁇ -lactamase system, the trp system, the tac and trc systems, the major operator and promoter regions of phage ⁇ , the con ⁇ trol region of fd coat protein, the early and late promoters of SV40, promoters derived from polyoma virus and adenovirus, etallothionine promoters, the promoter for 3-phosphoglycerate kinase or other gly- colytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast c * -mating fac ⁇ tors, and other sequences known to control the ex ⁇ pression of genes of prokaryotic or eukaryotic cells and their viruses or combinations thereof.
  • GM-CSF-Like Polypeptide A polypeptide displaying a biological activity of a GM-CSF. This polypeptide may include amino acids in addition to those of a mature GM-CSF or it may not include .all of the amino aqids of mature GM-CSF. Finally, it may include an N-terminal methionine.
  • the present invention relates to hGM-CSF- like polypeptides having a specific activity of at
  • this invention relates to the production of large amounts of hGM-CSF-like polypeptides in microbial cells.
  • the polypeptides of this invention are clinically useful as described previously; they also permit the produc- tion of both polyclonal and monoclonal antisera to human GM-CSF.
  • a wide variety of host/expression vehicle combinations may be employed in producing the GM-CSF-like polypeptides this invention in high ' yields.
  • a wide variety of host/expres ⁇ sion vehicle combinations may be employed to produce the high specific activity hGM-CSF-like polypeptides of this invention.
  • a deglycosylation step is, of course, required to produce the high specific acti ⁇ vity hGM-CSF of the present invention.
  • an appropriate host is controlled by a number of factors recognized by the art. These include, for example, compatibility with the chosen vector, toxicity of proteins encoded by the hybrid plasmid, ease of recovery of the desired protein, expression characteristics, bio-safety and cost. A balance of these factors must be struck with the understanding that not all host vector com- binations may be equally effective for the expression of the particular recombinant DNA molecules of this invention.
  • Useful expression vectors include, for example, vectors consisting of segments of chromo- somal, non-chromosomal and synthetic DNA sequences, such as various known derivatives of SV40, known bacterial plasmids, e.g., plasmids from E.coli including col El, pCRl, pBR322, pMB9 and their derivatives, wider host range plasmids, e.g., RP4, phage DNAs, e.g., the numerous derivatives of phage ⁇ , e.g., NM 989, and other DNA phages, e.g., M13 and Filêtous single stranded DNA phages, yeast plasmids such as the 2 ⁇ plasmid or derivatives thereof, and vectors derived from combinations of plasmids and phage DNAs, such as plasmids which have been modified to employ phage DNA or other expression control sequences.
  • vectors consisting of segments of chrom
  • any of a wide variety of expression control sequences sequences that con ⁇ trol the expression of a DNA sequence when opera- tively linked to it — may be used in these vectors to express the DNA sequence of this invention.
  • useful expression control sequences include, for example, the early and late promoters of SV40, the lac system, the trp system, the TAC or TRC system, the major operator and promoter regions of phage ⁇ , the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast c * -mating factors, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
  • the preferred expression vectors and con ⁇ trol sequences include the P.. promoter, the promoter of the yeast ⁇ -mating factor, and the yeast actin promoter.
  • host cells are also useful in producing the hGM-CSF-like polypeptides of this invention.
  • These hosts may include well known eukaryotic and prokaryotic hosts, such as strains of E.coli, Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animal cells, such as CHO cells, African green monkey cells, such as COS1, COS7, BSC1, BSC40, and BMT10, and human cells and plant cells in tissue culture.
  • Useful hosts may include strains of E.coli, such as E.coli W3110I ⁇ , E.coli JA221, E.coli C600, E.coli ED8767, E.coli DHl, E.coli LE392, E.coli HBlOl, E.coli X1776, E.coli X2282, E.coli MRCI, and strains of Pseudomonas, Bacillus, and Streptomyces, yeasts and other fungi, plant cells in culture or other hosts.
  • E.coli W3110I ⁇ E.coli JA221, E.coli C600, E.coli ED8767, E.coli DHl, E.coli LE392, E.coli HBlOl, E.coli X1776, E.coli X2282, E.coli MRCI
  • strains of E.coli such as E.coli W3110I ⁇ , E.coli JA221, E.coli C600, E.coli ED8767, E.coli DHl, E.coli
  • the preferred hosts of this invention include E.coli K 12 , E.coli strains SG927 [ATCC 39627], SG928 [ATCC 39628], SG935 [ATCC 39623] and SG936 [ATCC 39624] as well as its derivative, A89 [DSM 3869]., and yeast- strain Saccharo yces cerevisiae BJ1991.
  • the hGM-CSF-like polypeptides may include polypeptides in the form of fused proteins (e.g., linked to a prokaryotic, eukaryotic or combination N-terminal segment to direct excretion, improve stability, improve purification or improve possible cleavage of the N-terminal seg- ment), in the form of a precursor of GM-CSF-like polypeptides (e.g., starting with all or parts of a GM-CSF-like polypeptide signal sequence or other eukaryotic or prokaryotic signal sequences), in the form of a mature GM-CSF-like polypeptide, or in the form of a met-GM-CSF-like polypeptide.
  • fused proteins e.g., linked to a prokaryotic, eukaryotic or combination N-terminal segment to direct excretion, improve stability, improve purification or improve possible cleavage of the N-terminal seg- ment
  • One particularly useful form of a polypep ⁇ tide in accordance with this invention, or at least a precursor thereof, is a mature GM-CSF-like poly ⁇ peptide with an easily cleaved amino acid or series of amino .acids attached to the amino tei ⁇ inus.
  • Such construction allows synthesis of the protein in an appropriate host, where a translation start signal that may not be present in the desired GM-CSF is needed, and then cleavage in vivo or in vitro of the extra amino acids to produce the desired GM-CSF-like polypeptides.
  • the polypeptides of the invention also include hGM-CSF-like polypeptides that are coded for on expression by DNA sequences characterized by dif- ferent codons for some or all of the codons of the present DNA sequences. These substituted codons may code for amino acids identical to those coded for by the codons replaced but result in higher yield of the polypeptide. Alternatively, the replacement of one or a combination of codons leading to amino acid replacement or to a longer or shorter GM-CSF-like polypeptide may alter its properties in a useful way (e.g., increase the stability, increase the solu ⁇ bility or increase the therapeutic activity) . It should be understood that these polypeptides are also part of this invention.
  • hGM-CSFs of this invention are those characterized by a specific activity of at least 1 x 10 Units/mg.
  • These poly ⁇ peptides may be produced directly in hosts that do not glycosylate their polypeptides, e.g., bacterial hosts such as E.coli. They may also be produced by deglycosylating polypeptides produced in hosts that glycosylate their polypeptides, e.g., yeasts and animal cells. Such deglycosylation may be accom ⁇ plished either in vitro or in vivo using conven- tional methods and compositions well known in the art. It may also be accomplished by inhibiting glycosylation during protein production using con ⁇ ventional agents and methods.
  • the GM-CSF-like polypeptides of the pre- sent invention may be purified by a variety of con ⁇ vention steps and strategies. These methods are know in the art.
  • GM-CSF-like polypeptides of this invention may be a ' dministered in compositions and methods of treatment in the form in which they are produced, it should also be understood that they may be formulated using known methods to prepare pharma ⁇ ceutically useful compositions. Such compositions also will preferably include conventional pharma- ceutically acceptable carriers and may include other medicinal agents, carriers, adjuvants, excipients, etc., e.g., human serum albumin or plasma prepara ⁇ tions. See, e.g., Remington's Pharmaceutical Sciences (E. W. Martin).
  • the resulting formulations will contain an amount of hGM-CSF-like polypeptides effective in the recipient to stimulate the colony formation of granulocytes and macrophages.
  • Admini ⁇ stration of these polypeptides, or pharmaceutically acceptable derivatives thereof may be via any of the conventional accepted modes of administration of GM-CSF. These include parenteral, subcutaneous, or intravenous administration.
  • the GM-CSF-like polypeptides of this invention are particularly useful in compositions and methods for increasing the white blood cell count of immunologically-compromised patients so as to reduce the risk of infection in those patients.
  • the compositions and methods of this invention are useful in the therapy of AIDS patients- to prevent the occurrence of opportunistic infections which often shorten the life of the AIDS patient and add to the expense of their treatmen . *
  • the dosage and dose rate will depend on a variety of factors for example, whether the treatment is given to a cancer patient after radiation therapy or to an AIDS victim to prevent opportunistic infec ⁇ tion. However, the dosage will likely be between 1 and 10 ⁇ g per day or between 10 and 100 ⁇ g per week, if the patient is to be treated steadily over a long period of time.
  • RNA preparation was then enriched for poly(A) RNA by passage over an oligo(dT)-cellulose column (PL Biochem) . -15-
  • RNA was then added to a reaction mixture composed of 0.1 M Tris-HCl (pH 8.3) at 42°C, 0.01 M MgCl 2 , 0.01 M DTT, 1 mM dCTP, 1 mM dGTP, 1 mM dTTP, 0.5 mM dATP and 100 ⁇ Ci ⁇ - 32 P-ATP (3000 Ci/mmole, Amersham or New England Nuclear), 20 ⁇ g oligo ( d ⁇ ) 2 _i8 (** ?L B i° cnem )/ 0.03 M ⁇ -mercapto- ethanol, 5 mM Vanadyl Ribonucleoside Complex (Bethesda Research Labs), 169 U AMV Reverse Transcriptase
  • SM buffer (0.01 M Tris-HCl (pH 7.5), 0.01 M MgCl 2 , 0.1 mM Na 2 EDTA) and eluted on a gyro-rotary shaker overnight at 4°C.
  • the phage pellets were thoroughly drained, resuspended in 60 ml SM, and spun at 10,000 rpm in a SS34 rotor to remove debris. The supernatants were adjusted to 3.5 M CsCl by addition of 7 g CsCl to 10 ml supernatant. We obtained phage bands by cen ⁇ trifugation in a 70.1 Beckman rotor at 50,000 rpm for 18 h at 15°C. We pooled the phage bands and stored them at 4°C for library stock. The titer obtained was 2.2 x 10 PFU/ml. ! -19-
  • mouse GM-CSF cDNA might cross hybridize to human GM-CSF cDNA to an extent sufficient to allow selection of a human GM-CSF related cDNA from our human cDNA library.
  • the first 17 residues comprise a series of hydrophobic amino acids consistent with their role as putative a signal sequence for the remaining protein.
  • the cleavage site between serine (the terminal amino acid of the putative signal sequence) and alanine (the first amino acid of the coding sequence) as depicted in Figure 1, is also in agreement with the reported first amino acid of the mature protein (alanine).
  • the A to G substitution at position 297 results in a codon for- an isoleucine instead of the reported methionine.
  • FIG. 4 a schematic outline of one embodi- ent of a process for preparing a recombinant DNA molecule (pPLmuGM-CSF) characterized in that it pro ⁇ prises hGM-CSF in high yield. It has a U937-derived DNA sequence coding for human GM-CSF, which had been modified at 5' end of its coding region to minimize any potential disadvantageous RNA secondary structure, fused to a DNA sequence derived from mu and carrying a Shine Dalgarno sequence from mu, the combined DNA sequence being operatively-linked to a PL promoter dervied from bacteriophage ⁇ .
  • expression vector pPLmuGM-CSF To construct expression vector pPLmuGM-CSF, we first prepared a synthetic oligonucleotide DNA sequence or linker to replace the coding sequence between the first alanine codon and a unique Hga I site at nucleotide 120 of our hGM-CSF cDNA ( Figure 1).
  • This Ncol-Hgal linker ( Figure 6) was constructed to reduce the potential of mRNA secondary structure which might make the ribosomal binding site inacces ⁇ sible.
  • this linker we substituted adenosine (A) for the naturally occurring nucleotides wherever the degeneracy of the genetic code allowed the retention of the same amino acid sequence. [G. N.
  • pPLMU:hGM-CSF yielded significantly more hGM-CSF than p208 Bal31, proving that our synthetic linker and its inhibition of mRNA secondary structure markedly improved the production of hGM-CSF-like polypeptide.
  • thermosensitive respressor p210* into E.coli strain C600 which also carried a second plasmid encoding the thermosensitive respressor
  • E.coli strain SG936 [lac (am), trp (am), pho (am), sup C (ts), rpsl, mal (am), htpR (am), tsx:TN10, Ion R9] [ATCC 39624] which is an htpR Ion mutant.
  • This mutant is deficient -in its production of Ion protease.
  • this strain, as well as Ion mutant strains SG935 [ATCC 39623], SG927 [ATCC 39627], and SG928 [ATCC 39628] exhibits a reduced capacity to degrade foreign pro ⁇ teins upon their accumulation within the cell or at high temperatures.
  • One plasmid (210*) employed ampicillinase as its resistance marker. Unfortunately, the a picillin required in its growth medium is an undesirable element in fermentation for a human pharmaceutical product. In addition, coordinate growth of the two plasmids was not certain. Thus, the quantity of repressor encoded by one plasmid
  • the bacterial host strain (SG936) in the high expression system described above was also..
  • the cell preparation contained 7% blood cells, which proliferated in response to hGM-CSF [J. D. Griffin et al., Blood, 63, pp. 904-11 (1984)].
  • the cells (10 /ml) were incubated for 48 hours with varying concentrations of GM-CSF and proliferation was measured by a 6 hour incorporation
  • buffer I laOOmM sodium phosphate
  • phenylmethylsulfonyl fluoride 0.5 mM
  • the concentrated material was applied to a column of Ultrogel ACA-54 (LKB, 2.6 x 90 cm) equili- brated with 30 mM sodium phosphate and 130 mM NaCl
  • Figures 9 and 10 show a schematic outline of one embodiment of a process for constructing expres- sion vector p528/l which, when used to transform appropriate yeast cells, expressed hGM-CSF in high yields.
  • MF ⁇ l codes for a precursor of 165 amino acids, containing four copies of alpha factor.
  • the alpha factor repeats are preceded by a secretion leader sequence of 83 amino acids.
  • the junctions between the secretion leader and the first repeat, and between each of the repeats have the following structure:
  • the optimal cleavage of the secretion leader from the heterologous protein portion of the fusion pre ⁇ cursor may be obtained when the first amino acid of the heterologous protein is placed behind the -lys-arg processing site (see Figure 8). Accordingly, we used this alpha mating factor signal sequence hGM-CSF fusion in our vectors.
  • Gene 1 which was present on plasmid p210*, is characterized by a U937-derived DNA sequence coding for hGM-CSF, which was modified at the 5'-end of its coding region by a synthetic oligonucleotide DNA sequence or linker ( Figure 6).
  • Gene 2 which was present on plasmid p208 corresponds to unmodified hGM-CSF coding sequence.
  • KEX2 cleavage Recombinant plasmid p216 was cut with Eco RI and Hind III and we isolated the small fragment, which contained the MF ⁇ l/hGM-CSF fusion. This frag ⁇ ment was transferred to expression vector pl60/-I which carries an origin of replication for E.coli, the yeast URA 3 gene, the origin of replication of the 2 ⁇ circle, and the origin of replication of ARS 1 (autonomously replicating sequence, which allows replication in the yeast cell independently of the yeast chromosone), and the upstream region of PYK 1 (PUR) [D. T. Stinchomb et al., "Isolation and • Characterization Of A Yeast Chromosomal Replicator," Nature, 282, pp. 39-43 (1979)].
  • Absolute expression levels realized were as follows (measured at OD 600 10) in mg/litre: plasmid medium cells p525/2 10 5 p545/l 20 8 p528/l ⁇ 0.1 ⁇ 0.1
  • Native hGM-CSF is glycosylated; the hGM-CSF protein sequence contains two potential sites of N-linked glyco- sylation [Wong et al., Science, 228, pp. 810-15 (1985)].
  • the size of the 18 kd form was con ⁇ sistent with either the presence of two core- glycosyl side chains attached to each of both potential glycosylation sites, or the presence of an extended core glycosyl chain attached to only one glycosylation site.
  • (c) a high-molecular-weight glycosylated form (about 43 kd) .
  • This form comprised 80-90% of the total secreted hGM-CSF.
  • the supernatants from the cultures of yeast cells producing hGM-CSF were tested for biological activity.
  • yeast-secreted hGM-CSF stimulated colony-formation in a dose-dependent manner.
  • the CML assay described supra at pp. 24-25, showed a dose dependent response to yeast produced hGM-CSF.
  • Natural hGM-CSF is known to be a glycopro ⁇ tein with a molecular weight of about 22 kd [G.G. Wong et al., Science, 228, supra] .
  • the polypep ⁇ tide chain there are two asparagine residues at positions 27 and 37 which are potential sites for N-linked glycosylation (Asn-X-Thr/Ser) .
  • Asn-X-Thr/Ser N-linked glycosylation
  • Such deglycosylated hGM-CSFs may be pro ⁇ substituted in several ways. For example, they may be produced in bacterial cells that do not glycosylate the proteins they produce. For example, when the polypeptide chain is produced by E.coli, it contains no attached carbohydrate and possesses a molecular weight of 14.5 kd.
  • unglycosylated polypeptides may also be produced by deglycosylating yeast or animal cell produced proteins.
  • a high molecular weight fraction (MW 50-70 kd) of hGM-CSF produced in yeast cells, as described above, using ConA-chromatography and gel filtration.
  • hGM-CSF in animal cells, by growing transfected Chinese Hamster Ovary (CHO) cells for three days in 10% fetal calf -serum containing medium.
  • CHO-cell clone which was derived from transfection with a vector that had the hGM-CSF gene isolated from U937 cells. The transcription was promoted by a SV40 early and Adenovirus major late promoter.
  • Microorganisms and recombinant DNA mole ⁇ cules prepared by processes of this invention are exemplified by cultures deposited in the Deutsche Sammung von Mikroorganism, Grisebachstrasse 8 , D-3400 Gottingen, West Germany, on September 2 , 1985 and identified there as B84, B85 , B102 , and YE464 , and on October 4 , 1986 and identified there as Bill (p241-8 ) .
  • E.coli K ⁇ 2 (p210*-5637)

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  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Toxicology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Plant Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Polypeptides semblables aux stimulations de colonies de granulocytes-macrophages ayant une activité spécifique d'au moins 1 x 108 Unités/mg et leurs procédés de fabrication. Séquences d' ADN et molécules d'ADN recombinant et hôtes microbiens transformés par eux, qui produisent en quantités importantes des polypeptides semblables au facteur de stimulation de colonie de granulocyte-macrophage humaines, ainsi que des procédés de fabrication de ces polypeptides. Les polypeptides de cette invention semblables au facteur de stimulation de colonies de granulocytes-macrophages peuvent être utilisés pour le traitement des personnes souffrant d'un cancer pour régénérer les leucocytes après un traitement de radiations ou de chimiothérapie, et pour augmenter l'hémogramme des globules blancs afin de réduire la probabilité d'une infection virale, bactérielle, mycosique et parasitaire, spécialement dans le cas de patients dont l'immunologie est compromise, comme par exemple ceux qui sont atteints du SIDA.
PCT/US1986/002106 1985-10-03 1986-10-03 Polypeptides semblables aux facteurs de stimulation de colonies de granulocytes-macrophages (gm-csf) et leurs procedes de production en quantites importantes dans des cellules microbiennes WO1987002060A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
DK282887A DK282887A (da) 1985-10-03 1987-06-02 Humane granulocyt-makrofag-kolonistimulationsfaktor-lignende polypeptider og fremgangsmaader til fremstilling deraf i hoeje udbytter i mikrobielle celler

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US78341485A 1985-10-03 1985-10-03
US783,414 1985-10-03

Publications (1)

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WO1987002060A1 true WO1987002060A1 (fr) 1987-04-09

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PCT/US1986/002106 WO1987002060A1 (fr) 1985-10-03 1986-10-03 Polypeptides semblables aux facteurs de stimulation de colonies de granulocytes-macrophages (gm-csf) et leurs procedes de production en quantites importantes dans des cellules microbiennes

Country Status (5)

Country Link
EP (1) EP0238655A4 (fr)
JP (1) JPS63502795A (fr)
AU (2) AU606585B2 (fr)
DK (1) DK282887A (fr)
WO (1) WO1987002060A1 (fr)

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0276846A2 (fr) * 1987-01-29 1988-08-03 Zymogenetics, Inc. Dérivés du facteur stimulant des colonies
EP0281069A1 (fr) * 1987-03-02 1988-09-07 Sumitomo Chemical Company, Limited Production de facteur humain "colony stimulating" de granulocytes-macrophagues
EP0288809A1 (fr) * 1987-04-16 1988-11-02 Hoechst Aktiengesellschaft Protéines bifonctionnelles
EP0299782A2 (fr) * 1987-07-17 1989-01-18 Schering Biotech Corporation Vecteurs d'expression du GM-CSF humain dans des cellules de mammifères
WO1989003881A1 (fr) * 1987-10-30 1989-05-05 Immunex Corporation Analogues non glycosyles de facteurs humains de stimulation de colonies
GB2212159A (en) * 1987-11-13 1989-07-19 British Bio Technology Synthetic-gene
EP0340005A2 (fr) * 1988-04-27 1989-11-02 Immunomedics, Inc. Utilisation des cytokines pour l'amélioration de la radiothérapie
WO1990008554A1 (fr) * 1989-01-30 1990-08-09 Schering Corporation Traitement de dysfonctionnement leucocytaire a l'aide de gm-csf
US5104650A (en) * 1985-02-05 1992-04-14 Cetus Corporation Uses of recombinant colony stimulating factor-1
US5242811A (en) * 1985-03-26 1993-09-07 Biogen, Inc. Production of human somatomedin C
US5422105A (en) * 1985-02-05 1995-06-06 Cetus Oncology Corporation Use of recombinant colony stimulating factor 1
US5556620A (en) * 1985-02-05 1996-09-17 Cetus Oncology Corporation Use of recombinant colony stimulating factor-1 to enhance wound healing
US5837229A (en) * 1985-02-05 1998-11-17 Chiron Corporation Uses of recombinant colony stimulating factor-1
WO2003031464A2 (fr) 2001-10-10 2003-04-17 Neose Technologies, Inc. Remodelage et glycoconjugaison de peptides
US6979442B1 (en) 1998-08-17 2005-12-27 Pfizer Inc. Stabilized protein compositions
WO2010011735A2 (fr) 2008-07-23 2010-01-28 Ambrx, Inc. Polypeptides g-csf bovins modifiés et leurs utilisations
US10683355B2 (en) 2016-09-16 2020-06-16 Kissei Pharmaceutical Co., Ltd. Genetically-modified cells and method for producing same
US11273202B2 (en) 2010-09-23 2022-03-15 Elanco Us Inc. Formulations for bovine granulocyte colony stimulating factor and variants thereof

Families Citing this family (4)

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DE3545568A1 (de) * 1985-12-21 1987-07-16 Hoechst Ag Gm-csf-protein, seine derivate, herstellung solcher proteine und ihre verwendung
ATE121134T1 (de) * 1987-06-25 1995-04-15 Immunex Corp Rinder granulocyt-makrophagenkolonie stimulierender faktor.
JPH03503897A (ja) * 1988-04-21 1991-08-29 メドベット・サイエンス・プロプライアテリー・リミテッド ひとｇｍ‐ｃｓｆ変異形類
EP0791061A4 (fr) * 1994-03-04 1998-07-15 Ludwig Inst Cancer Res Animaux a rupture genique ciblee

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EP0188479B1 (fr) * 1984-07-06 1991-09-11 Sandoz Ag Production et purification de lymphokine
ZA856108B (en) * 1984-10-29 1986-10-29 Immunex Corp Cloning of human granulocyte-macrophage colony simulating factor gene
HU213226B (en) * 1984-11-20 1997-03-28 Schering Biotech Corp Method for production of cdna clones coding for polypeptides exhibiting human granulocyte macrophage and eosinophil cellular growth factor activity
US5391485A (en) * 1985-08-06 1995-02-21 Immunex Corporation DNAs encoding analog GM-CSF molecules displaying resistance to proteases which cleave at adjacent dibasic residues
DE3545568A1 (de) * 1985-12-21 1987-07-16 Hoechst Ag Gm-csf-protein, seine derivate, herstellung solcher proteine und ihre verwendung

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BLood, (New York), Volume 54, issued September, 1979 (NICOLA et al), "Separation of Functionally Distinct Human Granulocyte-Macrophage Colony Stimulating Factors", see pages 614-615. *
Nature (London, England), Volume 309, issued 28 June 1984, (GOUGH et al), "Molecular Cloning of cDNA Encoding a Murine Haematopoietic Growth Regulator, Granulocyte-Macrophage Colony Stimulating Factor", see page 763. *
Proceedings National Academy of Sciences (Washington, D.C. USA), issued June, 1984, (NICOLA et al), "Binding of the Differentiation Inducer, Granulocyte-Colony-Stiumlating Factor, to Responsive but not Unresponsive Leukemic Cell Lines", page 3865. *
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Science (Washington D.C. USA), Volume 228, issued May, 1985, (WONG et al), "Human GM-CSF:Molecular Cloning of the Complementary DNA and Purification of the Natural and Recominant Proteins", see pages 810 and 815. *
See also references of EP0238655A4 *
The EMBRO Journal, (Oxford, England), Volume 4, issued March, 1985, (GOUGH et al), "Structure and Expression of mRNA for Murine Granulocyte-Macrophage Colony Stimulating Factor", see page 645. *
The EMBRO Journal, (Oxford, England), Volume 4, issued October 1985, (MIYATAKE et al), "Structure of the Chromosomal Gene for Granulocyte-Macrophage Colony Stimulating Factor: Comparison of the Mouse and Human Genes", see page 2561. *

Cited By (33)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5104650A (en) * 1985-02-05 1992-04-14 Cetus Corporation Uses of recombinant colony stimulating factor-1
US5837229A (en) * 1985-02-05 1998-11-17 Chiron Corporation Uses of recombinant colony stimulating factor-1
US5725850A (en) * 1985-02-05 1998-03-10 Chiron Corporation Use of CSF-1 to treat tumor burden
US5635175A (en) * 1985-02-05 1997-06-03 Chiron Corporation Use of CSF-1 to treat viral infections
US5614183A (en) * 1985-02-05 1997-03-25 Chiron Corporation Use of CSF-1 to treat bacterial infections
US5556620A (en) * 1985-02-05 1996-09-17 Cetus Oncology Corporation Use of recombinant colony stimulating factor-1 to enhance wound healing
US5422105A (en) * 1985-02-05 1995-06-06 Cetus Oncology Corporation Use of recombinant colony stimulating factor 1
US5654177A (en) * 1985-03-26 1997-08-05 Biogen, Inc. Production of human somatomedin C
US5470721A (en) * 1985-03-26 1995-11-28 Biogen, Inc. Production of human somatomedin C
US5242811A (en) * 1985-03-26 1993-09-07 Biogen, Inc. Production of human somatomedin C
EP0276846A3 (fr) * 1987-01-29 1989-07-26 Zymogenetics, Inc. Dérivés du facteur stimulant des colonies
EP0276846A2 (fr) * 1987-01-29 1988-08-03 Zymogenetics, Inc. Dérivés du facteur stimulant des colonies
EP0281069A1 (fr) * 1987-03-02 1988-09-07 Sumitomo Chemical Company, Limited Production de facteur humain "colony stimulating" de granulocytes-macrophagues
EP0288809A1 (fr) * 1987-04-16 1988-11-02 Hoechst Aktiengesellschaft Protéines bifonctionnelles
EP0299782A2 (fr) * 1987-07-17 1989-01-18 Schering Biotech Corporation Vecteurs d'expression du GM-CSF humain dans des cellules de mammifères
EP0299782A3 (en) * 1987-07-17 1989-08-09 Schering Biotech Corporation Human granulocyte macrophage colony stimulating factor and muteins thereof
WO1989000582A3 (fr) * 1987-07-17 1989-02-23 Schering Biotech Corp Facteur stimulant la croissance des colonies de granulocytes-macrophages humains et ses muteines
WO1989000582A2 (fr) * 1987-07-17 1989-01-26 Schering Biotech Corporation Facteur stimulant la croissance des colonies de granulocytes-macrophages humains et ses muteines
WO1989003881A1 (fr) * 1987-10-30 1989-05-05 Immunex Corporation Analogues non glycosyles de facteurs humains de stimulation de colonies
GB2212159B (en) * 1987-11-13 1992-01-22 British Bio Technology Synthetic gene for human granulocyte/macrophage colony stimulating factor.
GB2212159A (en) * 1987-11-13 1989-07-19 British Bio Technology Synthetic-gene
EP0340005A3 (fr) * 1988-04-27 1991-06-12 Immunomedics, Inc. Utilisation des cytokines pour l'amélioration de la radiothérapie
EP0340005A2 (fr) * 1988-04-27 1989-11-02 Immunomedics, Inc. Utilisation des cytokines pour l'amélioration de la radiothérapie
US5178855A (en) * 1989-01-30 1993-01-12 Schering Corporation Treatment of luekocyte dysfunction with GM-CSF
WO1990008554A1 (fr) * 1989-01-30 1990-08-09 Schering Corporation Traitement de dysfonctionnement leucocytaire a l'aide de gm-csf
EP0382381A1 (fr) * 1989-01-30 1990-08-16 Schering Corporation Traitement des désordres leucocytaires par le GM-CSF
US6979442B1 (en) 1998-08-17 2005-12-27 Pfizer Inc. Stabilized protein compositions
WO2003031464A2 (fr) 2001-10-10 2003-04-17 Neose Technologies, Inc. Remodelage et glycoconjugaison de peptides
WO2010011735A2 (fr) 2008-07-23 2010-01-28 Ambrx, Inc. Polypeptides g-csf bovins modifiés et leurs utilisations
EP3225248A1 (fr) 2008-07-23 2017-10-04 Ambrx, Inc. Polypeptides g-csf bovins modifiés et leurs utilisations
US10138283B2 (en) 2008-07-23 2018-11-27 Ambrx, Inc. Modified bovine G-CSF polypeptides and their uses
US11273202B2 (en) 2010-09-23 2022-03-15 Elanco Us Inc. Formulations for bovine granulocyte colony stimulating factor and variants thereof
US10683355B2 (en) 2016-09-16 2020-06-16 Kissei Pharmaceutical Co., Ltd. Genetically-modified cells and method for producing same

Also Published As

Publication number Publication date
DK282887D0 (da) 1987-06-02
AU606585B2 (en) 1991-02-14
EP0238655A4 (fr) 1989-09-11
AU6578190A (en) 1991-01-10
DK282887A (da) 1987-07-30
EP0238655A1 (fr) 1987-09-30
AU6549386A (en) 1987-04-24
AU630516B2 (en) 1992-10-29
JPS63502795A (ja) 1988-10-20

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